The field of the invention is related in general terms to the field of tumor biology. In particular, the invention refers to selective-replication adenoviruses in tumors, known as oncolytic adenoviruses, and their use to inhibit cancer.
The current treatment of cancer is based principally on chemotherapy, radiotherapy, and surgery. Despite a high cure rate of cancer in early stages, the majority of advanced cases of cancer are incurable because they cannot be removed surgically or because the doses administered of radiotherapy or chemotherapy are limited because of their toxicity for normal cells. To alleviate this situation, biotechnological strategies have been developed that seek to increase the potency and selectivity of cancer treatments. Among these, gene therapy and virotherapy use viruses with the aim of treating cancer. In gene therapy, the virus is modified to prevent its replication and to act as a vehicle or vector of the therapeutic genetic material. On the other hand, virotherapy uses viruses that are replicated and propagated selectively in tumor cells1. In virotherapy, the tumor cell dies as a result of the cytopathic effect caused by the internal replication of the virus more than because of the effect of a therapeutic gene. Preferential replication in a tumor cell is called oncotropism and the lysis of the tumor is called oncolysis. Viruses that are replicated selectively in tumors are called oncolytic viruses.
Cancer virotherapy significantly predates gene therapy. The first observations of tumor cure with viruses date from early in the last century. Already in 1912, De Pace observed tumor regressions after inoculating the rabies virus in cervical carcinomata2. Since then, many types of virus have been injected in tumors to treat them3. There are viruses that present a natural oncotropism, for example the autonomous parvovirus, the vesicular-stomatitis virus5 and the reovirus6. Other viruses can be manipulated genetically for selective replication in tumors. For example, the herpes simplex virus (HSV) has been made oncotropic on selecting the gene of ribonucleotide reductase, a dispensable enzyme activity in cells in active proliferation such as tumor cells7. However, the adenovirus, in view of its low pathogenicity and high capacity to infect tumor cells, has been the virus used most in both virotherapy and gene therapy for cancer.
The type-5 human adenovirus (Ad5) is a virus formed by an icosahedral protein capsid that encloses a linear DNA of 36 kilobases8. In adults infection with Ad5 is usually asymptomatic, and in children it causes a common cold and conjunctivitis. In general, Ad5 infects epithelial cells, which during a natural infection are the cells of the bronchial epithelium. It enters the cells by means of interaction of the fiber, a viral protein that extends like an antenna from the twelve vertices of the capsid, with a cell protein involved in intercellular adhesion called Coxsackie-Adenovirus Receptor (CAR). When the viral DNA arrives inside the nucleus, methodical transcription of the early viral genes begins. The first viral genes expressed correspond to the genes of the early 1A (E1A) region. E1A bonds with an Rb cell protein that is forming a complex with the E2F transcription factor. Thus, E2F is released to activate the transcription of other viral genes such as E2, E3 and E4 and cell genes that activate the cell cycle. Also, E1B bonds with p53 to activate the cell cycle and prevent the apoptosis of the infected cell. E2 codifies for replication proteins of the virus, E3 for proteins that inhibit the antiviral immune response and E4 for proteins that transport viral RNA. The expression of these early genes leads to the replication of the viral DNA and once replicated, activates the promoter that regulates the expression of the late or structural genes that form the capsid.
Methods have been used to construct oncolytic adenoviruses: the selection of viral functions that are not necessary in tumor cells and the replacement of viral promoters with tumor-selective promoters1. In both strategies, the gene to be selected or regular gene belongs preferably to the E1 region, and in particular, affects E1a because it controls the expression of other viral genes. As for selections of viral functions, the protein E1b-55K has, for example, been eliminated. This protein inactivates p53 to induce in the infected cell the entry in phase S of the cell cycle and to prevent cell apoptosis. A mutated adenovirus in E1b-55K known as Onyx-015 has been used to treat tumors defective in p53 although with little clinical success owing to its low propagation capacity or oncolytic potency. Another mutation performed in the adenoviral genome to achieve selective replication in tumors affects the CR2 field of E1a. This E1a field mediates the bonding to proteins of the Retinoblastoma (Rb) family. pRb proteins block the transition of the Go/G1 phase to the S phase of the cell cycle, forming a complex transcription inhibitor along with E2F. When E1a bonds with a pRb, the E2F transcription factor of the pRb-E2F complex is released and E2F acts as a transcriptional activator of the genes responsible for moving on to the S phase and viral genes such as E2. The release of E2F is thus a key step in the replication of the adenovirus. In tumor cells, the cell cycle is out of control because pRb is absent or inactivated by hyperphosphorylation and E2F is free. In these cells, the inactivation of pRb by E1a is now not necessary. Thus, an adenovirus with a mutation in E1a called Delta-24 that prevents its bonding with pRb can be propagated normally in cells with inactive pRb9,10.
With regard to the strategy of replacing viral promoters with tumor-selective promoters, the E1a promoter has been replaced by various promoters such as the alpha-fetoprotein promoter, a prostatic-specific antigen (PSA), kallikrein, mucine 1 and osteocalcin11-15. However, a major problem has been identified in the use of cell promoters in the viral context: the existence of viral sequences that interfere with the proper regulation of the promoter and reduce selectivity16,17. It has been attempted to correct this loss of selectivity by regulating other viral genes as well as E1a, such as E1b, E2 and E418,19. The regulation of various viral genes can be done with a different promoter for each viral gene, for example the E2F1 promoter for E1a and the telomerase promoter for E4. In this case, the two promoters must be expressed at high levels to allow viral replication such that oncolytic potency can remain reduced in many tumor cells20. Alternatively, two viral genes can be regular with the same promoter, for example in the oncolytic adenovirus Onyx 411, in which E1a and E4 are regulated by the E2F1 promoter21. However, it has been demonstrated that the duplication of promoter sequences in the adenoviral genome causes genomic instability by recombination between these repeated sequences22. This problem is difficult to solve because any modification of the E4 region seems to cause genomic instability of the oncolytic adenovirus22. In addition, the transcriptional regulation of adenoviral genes is temporarily controlled such that E1a activates the expression of other early viral genes. This regulation is optimal for the viral cycle and is lost if the promoter of viral genes other than E1a is replaced by tumor-specific promoters. On the other hand, the problem of interference between viral sequences and the specific promoter used to control adenoviral replication is especially important when it is desired to regulate the transcription of E1a and E4, given that there are enhancers and localized origins of transcription in the terminal repetitions and in the adenovirus-packaging signal23-25. In the field of non-oncolytic vectors, this interference has been alleviated by the insertion between the promoter and these enhancers of isolating sequences derived from the HS4 locus of the B-globin gene of chickens26,27. The insulating mechanism of HS4 is based on the protein CTCF union which inhibits the interactions between factors present in the enhancer and the promoter28. This invention describes the use of an insulating sequence derived from the human genome in the context of the oncolytic adenovirus design.”
A particularly interesting promoter used in the design of oncolytic adenoviruses is the E2F1 promoter20,21,29,30. This promoter presents two E2F bonding sites. The family of E2F transcription factors regulates the transcription of genes that allow entry to the S phase of the cell cycle. These factors serve as activators when they are released and as repressors when they bond with the pRb retinoblastoma protein31. The bonding of pRb to E2F is regulated by phosphorylation of pRb such that the phosphorylation of pRb prevents its bonding with E2F. Tumors present alterations in the signal-translation routes that result in the hyperphosphorylation of pRb and an increase in free E2F. Thus, in tumors, genes are expressed that respond to E2F such as the E2F1 gene. On the other hand, in a normal quiescent cell, pRb is not phosphorylated and remains bonded to E2F, forming a complex that acts as a transcriptional repressor. In oncolytic adenoviruses, however, the simple regulation of E1a with the E2F1 promoter results in a low level of selective replication in tumors, of the order of 10 times20. The regulation of other viral genes in addition to E1a is a possible solution to this low selectivity, but presents the problems described in the paragraph above. For example, OAS403 is an oncolytic adenovirus with E1a regulated with the promoter of E2F1 and E4 regulated with the promoter of telomerase, which furthermore includes a polyadenylation signal to eliminate transcription from the ITR (inverted terminal repetition) and in which the packaging signal has been relocated to the extreme right of the genome to reduce interference with the E1a promoter20. During the amplification of OAS403, it has been seen that the packaging signal and sequences adjacent to E4 change position in the genome22. It has moreover been described that even minor modifications of the E4 region cause genomic instability, and so strategies based on modification of the E4 region have been abandoned22. Another problem found with the E2F1 promoter apart from its selectivity is the lack of potency. In addition to being not very selective, an oncolytic adenovirus with E1a regulated by the E2F1 promoter loses its lytic capacity with regard to the salvage adenovirus as shown by Ryan et al.20 and in the examples presented in this invention.
This invention describes the use of appropriate DNA sequences to achieve the correct functioning of a genome promoter of an oncolytic adenovirus. With these sequences, an oncolytic adenovirus is designed that presents greater selectivity and anti-tumor potency. The use of the elements described in this invention allows the attainment of a high tumor selectivity and oncolytic capacity using only a tumor-specific promoter. The use of a single promoter reduces the problems of genomic instability associated with the repetition of the same promoter in the adenoviral genome. In addition, the regulation of only E1a, avoiding the regulation of other viral genes, allows the correct temporal regulation of adenoviral genes and prevents the genomic instability associated with modification of the E4 region.